Method for Production of Arachidonic Acid

ABSTRACT

The present invention relates to a process for producing arachidonic acid. In one embodiment,  Mortierella  sect.  schmuckeri  microorganisms are cultured in fermentation medium, preferably containing a component of a complex nitrogen source. Further disclosed is a food product which includes  Mortierella  sect.  schmuckeri  microorganisms or lipid isolated from such microorganisms to enhance the arachidonic acid content of the food product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/531,439, filed Sep. 13, 2006, which is a continuation of U.S. patentapplication Ser. No. 10/802,392, filed Mar. 4, 2004, now U.S. Pat. No.7,195,791, which is a continuation of U.S. patent application Ser. No.09/972,550, filed Oct. 5, 2001, now U.S. Pat. No. 6,749,849, which is adivisional of U.S. patent application Ser. No. 09/480,060, filed Jan.10, 2000, now U.S. Pat. No. 6,319,698, which is a continuation of U.S.patent application Ser. No. 09/270,294, filed Mar. 15, 1999, now U.S.Pat. No. 6,245,365, which is a continuation of U.S. patent applicationSer. No. 08/763,973, filed Dec. 10, 1996, now U.S. Pat. No. 5,882,703,which is a divisional of U.S. patent application Ser. No. 08/377,766,filed Jan. 24, 1995, now U.S. Pat. No. 5,583,019. The entire disclosureof U.S. patent application Ser. No. 09/972,550 is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a process for production of arachidonicacid. The present invention also relates to a food product and a methodto make the food product containing such arachidonic acid.

BACKGROUND OF THE INVENTION

Arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid) is apolyunsaturated fatty acid (PUFA) containing 20 carbon atoms with fourdouble bonds. The double bonds are arranged with the last one locatedsix carbon atoms from the methyl end of the chain. Therefore,arachidonic acid is referred to as an omega-6 fatty acid. Arachidonicacid is one of the most abundant C₂₀ PUFA's in the human body. It isparticularly prevalent in organ, muscle and blood tissues. Arachidonicacid is a direct precursor for a number of circulating eicosenoids, suchas prostaglandins, thromboxanes, leukotrienes and prostacyclins, whichare important biological regulators. These eicosenoids exhibitregulatory effects on lipoprotein metabolism, blood rheology, vasculartone, leukocyte function, platelet activation and cell growth. Theapplication of arachidonic acid to an infant's diet is particularlyimportant due to the rapid body growth of an infant. Arachidonic acid isan important precursor to many of the eicosanoids which regulatecellular metabolism and growth in infants. It is found naturally inhuman breast milk but not in most infant formula. In an effort to haveinfant formula match the long chain fatty acid profile found in breastmilk, scientific and food regulatory bodies have recommended thatarachidonic acid be added to infant formula, especially in formulautilized for premature infants.

In particular, it is preferable that arachidonic acid containing oilproduced for use with infant formula contain little or no other longchain highly unsaturated fatty acids (e.g., eicosapentanoic acid). Suchother long chain highly unsaturated fatty acids are not preferredbecause some of these fatty acids can interfere with the utilization ofarachidonic acid by the infant, and/or can inhibit blending of thearachidonic acid-containing oil with other oils to achieve theappropriate ratio of fatty acids matching breast milk or other desiredapplications. Highly unsaturated fatty acids are defined as fatty acidscontaining 4 or more double bonds.

Traditional sources of arachidonic acid include poultry eggs, bovinebrain tissue, pig adrenal gland, pig liver and sardines. The yield ofarachidonic acid, however, is usually less than 0.2% on a dry weightbasis. The use of microorganisms capable of producing arachidonic acidde novo have been suggested by various investigators, including Kyle,PCT Publication No. WO 92/13086, published Aug. 6, 1992; Shinmen et al.,U.S. Pat. No. 5,204,250, issued Apr. 20, 1993; Shinmen et al., pp.11-16, 1989, Appl. Microbiol. Biotechnol., vol. 31; Totani et al., pp.1060-1062, 1987, LIPIDS, vol. 22; Shimizu et al., pp. 509-512, 1992,LIPIDS, vol. 27; Shimizu et al., pp. 342-347, 1989, JAOCS, vol. 66;Shimizu et al., pp. 1455-1459, 1988, JAOCS, vol. 65; Shimizu et al., pp.254-258, 1991, JAOCS, vol. 68; Sajbidor et al., pp. 455-456, 1990,Biotechnology Letters, vol. 12; Bajpai et al., pp. 1255-1258, 1991,Appl. Environ. Microbiol., vol. 57; Bajpai, pp. 775-780, 1991, JAOCS,vol. 68; and Gandhi et al., pp. 1825-1830, 1991, J. Gen. Microbiol.,vol. 137. The arachidonic acid productivity by the microorganismsdisclosed by prior investigators, however, is less than 0.67 grams perliter per day. Such amounts are significantly less than the amounts ofarachidonic acid produced by the microorganisms of the presentinvention. These lower productivity values are the result of employingstrains: (1) with slow growth or lipid production rates leading to longfermentation times (i.e., greater than 2-3 days) (Kyle, 1992, ibid.;Shinmen et al., 1993, ibid.; Shinmen et al., 1989, ibid.; Bajpai et al.,1991, ibid.; Bajpai, ibid.; and Gandhi et al., ibid.); and/or (2) thatcontain low arachidonic acid contents (expressed as % fatty acids) inthe final oil produced (Shinmen et al., 1993, ibid.; Shimizu et al.,1989, ibid.; and Kendrick and Ratledge, 1992, pp. 15-20, Lipids, vol.27); and/or (3) which require long periods of stress (i.e., aging abiomass for 6-28 days) to achieve high levels of arachidonic acid in abiomass (Bajpai et al., 1991, ibid. and Shinmen et al., 1989, ibid.);and/or (4) that only exhibit high arachidonic acid content innon-commercial growth conditions (e.g., malt agar plates) (Totani andOba, 1987, pp. 1060-1062, Lipids, vol. 22). In addition, non-Mortierellaschmuckeri microorganisms that have been proposed for producingarachidonic acid, in particular Pythium insidiosum microorganisms,disclosed by prior investigators (Kyle, 1992, ibid.), have been reportedto be pathogenic to humans and/or animals.

Thus, there remains a need for an economical, commercially feasiblemethod for producing arachidonic acid. The present invention satisfiesthat need. There also remains a need for the an economical, commerciallyfeasible food product for the introduction of arachidonic acid producedaccording to the present invention into the diet of human infants.

SUMMARY

The present invention provides for a method for economically producingarachidonic acid. One embodiment of the present invention includes amethod to produce arachidonic acid, comprising culturing microorganismsof the genus Mortierella sect. schmuckeri in a medium comprising asource of assimilable organic carbon and a source of assimilablenitrogen. In another embodiment, such strains of Mortierella sect.schmuckeri are capable of producing at least about 0.86 grams per literper day of arachidonic acid.

Yet another embodiment of the present invention includes a food productcomprising lipids recovered from a microorganism of the genusMortierella sect. schmuckeri and a food material. In particular, suchlipids can be added to infant formula and baby food to increase thearachidonic acid or long chain omega-6 fatty acid content of such foods.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a novel process for the production ofcommercially feasible amounts of arachidonic acid using a Mortierellasect. schmuckeri microorganism. One embodiment of the present process isto produce arachidonic acid by culturing microorganisms of the genusMortierella sect. schmuckeri in a medium comprising a source ofassimilable organic carbon and a source of assimilable nitrogen. Thelower fungi Phycomycetes contains at least two classes, includingOomycetes and Zygomycetes. The class Zygomycetes contains at least twoorders, including Entomophthorales and Mucorales. Contained within theMucorales order are numerous genera including Mortierella.

The genus Mortierella contains nine sections, including sect. schmuckeri(Gams, 1977, pp. 381-391, Persoonia, vol. 9 and Gams, 1977, p. 216, inAbstracts Vol. A-L, Second International Mycological Congress,University of South Florida). The schmuckeri sect. of the genusMortierella contains three species referred to an Mortierellacamargensis, Mortierella clausenii and Mortierella schmuckeri.

All of the other strains of Mortierella that have been evaluated forarachidonic acid production belong to the Mortierella sections alpina,hygrophila or spinosa. It has now been recognized that strains ofMortierella sect. schmuckeri are particularly advantageous in theproduction of arachidonic acid compared to these other strains ofMortierella. In particular, it has been found that strains ofMortierella sect. schmuckeri are capable of producing arachidonic acidwith high productivity. Strains of Mortierella sect. schmuckeri of thepresent invention are preferably capable of producing at least about0.70 grams of arachidonic acid per liter per day, more preferably atleast about 0.80 grams of arachidonic acid per liter per day, and evenmore preferably at least about 0.86 grams of arachidonic acid per literper day. Preferably, strains of Mortierella sect. schmuckeri of thepresent invention are also capable of producing a total fatty acidcontent of at least about 20% of dry weight, preferably at least about30% of dry weight, and more preferably at least about 40% of dry weight.Moreover, preferred strains of Mortierella sect. schmuckeri of thepresent invention contain at least about 20% of total fatty acids asarachidonic acid, more preferably at least about 35% total fatty acidsas arachidonic acid, and even more preferably at least about 48% totalfatty acids as arachidonic acid. The arachidonic acid content ofcellular biomass of strains of Mortierella sect. schmuckeri of thepresent invention can be at least about 5% of cellular dry weight,preferably at least about 8% of cellular dry weight, and more preferablyat least about 13% of cellular dry weight.

Oil recovered, such as by extraction, from a preferred strain ofMortierella sect. schmuckeri of the present invention contains at leastabout 20% arachidonic acid, more preferably at least about 30%arachidonic acid, and even more preferably at least about 41%arachidonic acid. As used herein, “lipid” “lipid extract”, “oil” and“oil extract” are used interchangeably.

Morphological growth forms of fungi can have a significant effect ongrowth and product formation in fermenters. Fungal morphology infermenters can range from a dispersed filamentous form to a dense pelletform. Species of Mortierella sect. schmuckeri of the present inventionhave an advantage over previously utilized species of Mortierella,including the ability to readily grow (early in a fermentation) in adispersed filamentous form when grown in agitated liquid cultures suchas shake flasks or fermenters. Some other species of Mortierella grownin fermentation medium typically grow in the form of pellets orspherical aggregates (i.e., having the appearance of a very tight cottonball), sometimes exhibiting a dispersed form only after several days ina fermentation. Without being bound by theory, it is believed that thegrowth and productivity of cells in the pellet form is limited becausecells in the center of a pellet or aggregate are not exposed to thenecessary nutrients contained in the fermentation medium. Traditionalmethods of growing these fungal populations can include increasing theagitation of the fermenter or addition of detergents in an attempt todisperse such aggregates and improve cell growth. The present inventorhas discovered that strains of Mortierella sect. schmuckeri of thepresent invention readily grow in the dispersed filamentous form,thereby improving growth and productivity of such cells by enablingnutrients to reach all the cells. As used herein, the term “filamentous”refers to the growth of fungi as a loosely branched network of shortmycelia rather than as a pellet or aggregate.

Preferred strains of Mortierella sect. schmuckeri of the presentinvention include strains of Mortierella sect. schmuckeri isolated fromcold, arid soil, in which the microorganisms experience short periods ofwetness. In particular, such areas can include soils that experiencesome prolonged periods of freezing or near freezing conditions. Morepreferred strains of Mortierella sect. schmuckeri are isolated from theSouthwest region of North America, in particular, desert regions of theUnited States and/or Mexico. In particular, strains of Mortierella sect.schmuckeri of the species Mortierella schmuckeri are isolated fromsouthern California and/or Mexico.

Strains of Mortierella can be isolated from soils or aquatic habitatsusing techniques known in the art (Stevens, 1974, in Mycology Guidebook,University of Washington Press, Seattle; and Barron, pp. 405-427, 1971,in Methods of Microbiology, Vol. 4.). More specifically, species ofMortierella sect. schmuckeri can be isolated by suspending small samplesof soil in distilled water and then streaking a portion of thesuspension on corn meal agar plates or agar plates containing a desiredfermentation media. Additionally, species of Mortierella sect.schmuckeri can be isolated from aquatic habitats using techniques knownin the art (see, for example, U.S. Pat. No. 5,130,242, by Barclay etal., issued Jul. 14, 1992; and U.S. Pat. No. 5,340,594, by Barclay etal., issued Aug. 23, 1994). On agar plates, Mortierella colonies can bepartly identified by several characteristics, including for example, aswhite colored colonies which grow essentially within the agar ratherthan predominantly exhibiting aerial growth. Mortierella colonies canalso be distinguished from other fungi using the general characteristicsof fungal taxonomy outlined, for example, by Talbot (Principles ofFungal Taxonomy, 1971, Macmillan Press). After isolation of a purecolony, members of the genus Mortierella can also be identified by, forexample, a garlic-like odor when cultured in a shake flask or in agarplate cultures containing media described in Stevens, ibid. The cultureproducing the best sporulation can then be used to identify the speciesof the culture using the Mortierella keys outlined in Gams (pp. 381-391,1977, Persoonia, Vol. 9; and in Taxonomic problems in Mortierella,Abstracts, 2nd International Mycological Conference, University of SouthFlorida, Tampa, published by Hamilton Newell, Inc., Amherst, Mass.).

After isolation of a pure colony of strains of Mortierella sect.schmuckeri, the biomass of the strain can be analyzed for lipid contentand arachidonic acid content by gas chromatography. Preferred coloniesthat exhibit rapid growth and high lipid and high arachidonic acidcontent can then be selected. Further selection for the presence orabsence of other characteristics can also be conducted. For example, inthe application of extracted lipids in infant formula for the benefit ofarachidonic acid content, the presence of eicosapentanoic acid(C20:5n-3; “EPA”) is detrimental. Therefore, one can select for theabsence of high EPA content.

One preferred species of Mortierella sect. schmuckeri of the presentinvention is Mortierella camargensis. Particularly preferred strains ofMortierella camargensis of the present invention have the identifyingcharacteristic of being able to produce about 0.86 grams of arachidonicacid per liter per day. Another identifying characteristic is thatbetween about 25% and about 33% of the total fatty acids produced bysuch particularly preferred Mortierella camargensis can be arachidonicacid. Thus, the resulting arachidonic acid content of a biomass of aparticularly preferred Mortierella camargensis of the present inventioncan be between about 9.6% and about 10.8% under appropriate fermentationconditions. Yet another identifying characteristic is that the resultingoil recovered from a particularly preferred Mortierella camargensis ofthe present invention can have an arachidonic acid content ranging fromabout 20% to about 30% of the total fatty acids.

Another particularly preferred species of Mortierella sect. schmuckeriof the present invention, Mortierella schmuckeri has the identifyingcharacteristic of being able to produce about 0.84 grams of arachidonicacid per liter per day. Another identifying characteristic is thatbetween about 40% and about 49% of the total fatty acids produced bysuch particularly preferred Mortierella schmuckeri of the presentinvention can be arachidonic acid. Thus, the resulting arachidonic acidcontent of a biomass of a particularly preferred Mortierella schmuckeriof the present invention can be between about 12.5% and about 13.6%under appropriate fermentation conditions. Yet another identifyingcharacteristic is that the resulting oil recovered from a particularlypreferred Mortierella schmuckeri can have an arachidonic acid contentranging from about 33% to about 41% of the total fatty acids.

It is within the scope of the present invention that, in addition toknown strains of Mortierella sect. schmuckeri, such as those on depositwith the American Type Culture Collection (e.g., ATCC), newly identifiedstrains from nature and mutant strains derived from known or newlyidentified strains, can be used to produce arachidonic acid.Naturally-occurring mutants of a parental strain of Mortierella sect.schmuckeri that are capable of producing arachidonic acid can beisolated by, for example, subjecting a parental strain to at least oneround of chemical or physical mutagenesis in order to increase the rateof mutagenesis, thereby increasing the probability of obtaining amicroorganism producing increased amounts of arachidonic acid. It willbe obvious to one skilled in the art that mutant microorganisms of thepresent invention also include arachidonic acid-producing microorganismsthat can be obtained by genetically-engineering microorganisms toproduce increased amounts of arachidonic acid. For example, it is withinthe purview of the present invention to transform Mortierella sect.schmuckeri microorganisms with nucleic acid molecules encoding enzymesof the arachidonic acid biosynthetic pathway obtained from fungalarachidonic acid-producing microorganisms, such as those of the genusMortierella sect. schmuckeri. A Mortierella sect. schmuckeri nucleicacid molecule of the present invention can be obtained from its naturalsource either as an entire (i.e., complete) gene or a portion thereofcapable of forming a stable hybrid with the entire gene. A nucleic acidmolecule from a strain of Mortierella sect. schmuckeri can also beproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. As usedherein, a “mutated microorganism” is a mutated parental microorganism inwhich the nucleotide composition of such microorganism has been modifiedby mutation(s) that occur naturally, that are the result of exposure toa mutagen, or that are the result of genetic engineering.

Preferred mutants of strains of Mortierella sect. schmuckeri of thepresent invention have one or more of the identifying characteristics ofa preferred Mortierella camargensis of the present invention and apreferred Mortierella schmuckeri of the present invention as describedin detail above.

In accordance with the present invention, microorganisms of the genusMortierella sect. schmuckeri capable of producing arachidonic acid, arecultured in an effective medium, herein defined as any medium capable ofpromoting arachidonic acid production. Preferably, the effective mediumalso promotes rapid fungal growth. The microorganisms of the genusMortierella sect. schmuckeri of the present invention can be cultured inconventional fermentation modes, which include, but are not limited to,batch, fed-batch, and continuous.

The present invention provides a method to produce arachidonic acid,comprising culturing microorganisms of the genus Mortierella sect.schmuckeri in a medium comprising a source of assimilable organic carbonand a source of assimilable nitrogen.

Sources of assimilable carbon include but are not limited to sugars andtheir polymers, including starches, dextrin, saccharose, maltose,lactose, glucose, fructose, mannose, sorbose, arabinose, xylose,levulose, cellobiose, and molasses; fatty acids; and polyalcohols suchas glycerine. Preferred carbon sources in the present invention includemonosaccharides, disaccharides, and trisaccharides. The most preferredcarbon source is glucose.

Sources of assimilable nitrogen useful for fermentation of amicroorganism of the present invention include simple nitrogen sources,organic nitrogen sources ad complex nitrogen sources. Such nitrogensources include ammonium salts and substances of animal, vegetableand/or microbial origin. Such organic nitrogen sources include cornsteep liquor, protein hydrolysates, microbial biomass hydrolysates, soytone, soy meal, fish meal, meat meal, meat extract, peptone, tryptone,yeast extract, yeast, whey, ammonium sulfate, urea, ammonium nitrate andamino acids.

Preferred nitrogen sources for use in an effective medium of the presentinvention include complex nitrogen sources. Use of a complex nitrogensource in a fermentation medium of the present invention increasesarachidonic acid production by a strain of Mortierella sect. schmuckeriof the present invention by at least about 50 percent and preferably byat least about 100 percent, either as measured by percent dry weight orpercent total fatty acids in an oil, compared with a strain ofMortierella sect. schmuckeri grown in the absence of a complex nitrogensource. Suitable complex nitrogen sources include, for example, cornsteep liquor, protein hydrolysates, microbial biomass hydrolysates, soytone, soy meal, fish meal, meat meal, meat extract, peptone, tryptone,yeast extract, yeast and whey. One of skill in the art can determinewhich complex nitrogen source best stimulates arachidonic acidproduction in the strain of Mortierella sect. schmuckeri employed in afermentation process.

In a preferred embodiment of the present invention, a fermentation isconducted in which a non-carbon nutrient, for example, nitrogen ormagnesium and preferably nitrogen, is limited. In this manner, cellularmetabolism is directed towards lipid production, thus enhancing theoverall production of arachidonic acid.

The effective medium can contain other compounds such as inorganicsalts, vitamins, trace metals or growth promoters. Such compounds can bepresent in carbon, nitrogen, or mineral sources in the effective mediumor can be added specifically to the medium. Low concentrations ofmagnesium are also preferred.

During the fermentation, variables including the oxygen content, pH,temperature, carbon dioxide content, and rate of carbon source additionare controlled to maximize the production of arachidonic acid withoutunduly limiting the length of time during which successful fermentationcan be accomplished. The optimum oxygen concentration for arachidonicacid production can be determined for any particular population ofMortierella sect. schmuckeri by variation of the oxygen content of themedium. In particular, the oxygen content of the fermentation medium ismaintained at an oxygen content preferably ranges from between about 20%of saturation and about 60% of saturation.

Growth of strains of Mortierella sect. schmuckeri of the presentinvention can be effected at any temperature conducive to satisfactorygrowth of the strain; for example, between about 25° C. and about 33°C., preferably between about 27° C. and about 32° C., and morepreferably at about 30° C. The culture medium typically becomes morealkaline during the fermentation if pH is not controlled by acidaddition or buffers. The strains of Mortierella sect. schmuckeri of thepresent invention will grow over a pH range from between about 4.0 toabout 10.0 with a starting pH of about 5.5 being more preferred.

Another aspect of the present invention includes a food productcomprising a food material combined with microorganisms of the genusMortierella sect. schmuckeri. Strains of Mortierella sect. schmuckeri ofthe present invention are added to a food material to create a foodproduct having enhanced concentrations of arachidonic acid. As usedherein, the term “food material” refers to any food type fed to humansor non-human animals. Also within the scope of the present invention isa method to make a food product comprising adding microorganisms of thegenus Mortierella sect. schmuckeri to a food material.

Mortierella sect. schmuckeri of the present invention are recovered foruse as a food supplement simply by separating the cells fromfermentation medium. A variety of procedures can be employed in therecovery of microbial cells from the culture medium. In a preferredrecovery process, the cells produced in the fermentation process arerecovered from the culture medium by separation using conventionalmeans, such as centrifugation or filtration. The cells can then bewashed, frozen, lyophilized, and/or dried (e.g., spray drying, tunneldrying, vacuum drying, or a similar process). The arachidonic acid richoil can be extracted immediately from the cells or the resulting cellscan then be stored under a non-oxidizing atmosphere of a gas such as N₂or CO₂ (to eliminate the presence of O₂) prior to incorporation into afood material. Alternatively, recovered cells can be used directly(without drying) as a feed supplement. To extend its shelf life, the wetbiomass of a strain of Mortierella sect. schmuckeri can be acidified(approximate pH=3.5-4.5) and/or pasteurized or flash heated toinactivate enzymes and then canned, bottled or packaged under a vacuum.

A suitable food material useful for the formation of a food product ofthe present invention includes animal food. The term “animal” means anyorganism belonging to the kingdom Animalia and includes, withoutlimitation, primates (e.g., humans and monkeys), livestock and domesticpets. The term “food product” includes any product to be fed to suchanimals. Preferred food materials to be consumed by humans includesinfant formula and baby food. Preferred food materials to be consumed bydomestic pets includes dog foods. By adding Mortierella sect. schmuckeribiomass or extracted oil to provide a source of arachidonic acid,preferred food products of the present invention comprise a total fattyacid content in which up to about 20% by weight of total fatty acids isarachidonic acid, more preferred food products of the present inventioncomprise a total fatty acid content in which up to about 10% by weightof total fatty acids is arachidonic acid, and even more preferred foodproducts of the present invention comprise a total fatty acid content inwhich between about 0.1% and about 1.0% by weight of total fatty acidsis arachidonic acid.

A further embodiment includes a food product comprising lipids recoveredfrom a microorganism of the genus Mortierella sect. schmuckeri and afood material. Recovered lipids can include either all of the lipidsrecovered from the microorganisms or a portion thereof (i.e., isolatedarachidonic acid or total fatty acids containing arachidonic acid). Inthe former instance, the lipid composition includes arachidonic acid inabout the same relative amount as it exists in the organism.Alternatively, the recovered lipids can be further processed toconcentrate the arachidonic acid to achieve a composition having agreater concentration of arachidonic acid than occurs naturally in theorganism. Also within the scope of the present invention is a method tomake a food product comprising adding lipids recovered from amicroorganism of the genus Mortierella sect. schmuckeri to a foodmaterial.

Recovery of lipids from strains of Mortierella sect. schmuckeri can beaccomplished by any suitable method, including numerous methods known inthe art. For example, recovery can include the following method.Harvested cells (fresh or dried) can be ruptured using techniques knownto those in the art. Lipids can then be extracted from the cells by anysuitable means, such as by supercritical fluid extraction, or byextraction with solvents such as chloroform, hexane, methylene chloride,methanol, isopropol, ethyl acetate, and the like, and the extractevaporated under reduced pressure to produce a sample of concentratedlipid material. Arachidonic acid can be further separated from otherlipids by chilling a fatty acid composition such that the saturatedfatty acids in the composition precipitate out while the arachidonicacid remains in solution. The solution can then be recovered.

The Mortierella sect. schmuckeri microorganisms can also be broken orlysed and the lipids recovered into edible oil using standard methodsknown in the art. The recovered oils can be refined by well-knownprocesses routinely employed to refine vegetable oils (e.g., chemical orphysical refining). These refining processes remove impurities fromrecovered oils before they are used or sold as edible oils. The refiningprocess consists of a series of processes to degum, bleach, filter,deodorize and polish the recovered oils. After refining, the oils can beused directly as a feed or food additive to produce arachidonic acidenriched products. Alternatively, the oil can be further processed andpurified as outlined below and then used in the applications asdescribed herein.

Lipids recovered from the biomass of a strain of Mortierella sect.schmuckeri of the present invention can be combined with any animal foodmaterial, particularly food materials for humans, to create a foodproduct having enhanced concentrations of arachidonic acid. The amountof fatty acids naturally in food products varies from one food productto another. A food product of the present invention can have a normalamount of arachidonic acid or a modified amount of arachidonic acid. Inthe former instance, a portion of the naturally occurring lipids aresubstituted by lipids of the present invention. In the latter instance,naturally occurring lipids are supplemented by lipids of the presentinvention.

Preferably, lipids recovered from strain of Mortierella sect. schmuckeriare added to foods for infants, such as infant formula and baby food.According to the present invention, an infant refers to infants in uteroand children less than about two years old, including, in particular,premature infants. Arachidonic acid is a particularly importantcomponent of infant formula and baby food because of the rapid growth ofinfants (i.e., doubling or tripling in weight during the first year oflife). An effective amount of arachidonic acid to supplement infantformula is an amount that approximates the concentration of arachidonicacid in human breast milk. Preferred amounts of arachidonic acid to addto infant formula or baby food range from between about 0.1 to about1.0% of total fatty acids, more preferably from between about 0.1 toabout 0.6% of total fatty acids, and even more preferably about 0.4% oftotal fatty acids.

Arachidonic acid produced by the method of the present invention issuitable for use as therapeutic and experimental agents. An embodimentof the present invention comprises the production of arachidonic acidfor treatment of arachidonic acid-deficient infants. The arachidonicacid can be included in a parenteral formulation that can beadministered to an infant through parenteral routes to fortify theinfant's supply of arachidonic acid. Preferred parenteral routesinclude, but are not limited to, subcutaneous, intradermal, intravenous,intramuscular and intraperitoneal routes. A parenteral formulation caninclude arachidonic acid of the present invention and a carrier suitablefor parenteral delivery. As used herein, a “carrier” refers to anysubstance suitable as a vehicle for delivering a molecule or compositionto a suitable in vivo site of action. Examples of such carriers include,but are not limited to water, phosphate buffered saline, Ringer'ssolution, dextrose solution, serum-containing solutions, Hank's solutionand other aqueous physiologically balanced solutions. Acceptableprotocols to administer arachidonic acid in an effective manner includeindividual dose size, number of doses, frequency of dose administration,and mode of administration. Determination of such protocols can beaccomplished by those skilled in the art depending upon a variety ofvariables, including the weight of the infant and the extent ofarachidonic acid deficiency. Another embodiment of the present inventioncomprises the production of arachidonic acid for treatment of adults, inparticular pregnant mothers. Acceptable protocols for administration ofarachidonic acid to adults includes parenteral feeding techniques orencapsulating oil recovered from a microorganism of the presentinvention in a capsule, such as gelatin (i.e., digestible) capsule, fororal administration and/or in a liquid diet formulation. A liquid dietformulation can comprise a liquid composition containing nutrientssuitable for supplementing a diet or nutrients sufficient as a completediet.

Another embodiment of the present invention comprises the production ofarachidonic acid for use as an experimental reagent to identifyregulators of metabolic pathways for which arachidonic acid is aprecursor. For example, arachidonic acid is a precursor forleukotrienes. Leukotrienes are believed to be involved in the occurrenceof certain diseases involving inflammation and allergy. As such,inhibitors of leukotriene production may be valuable therapeutic agents.Arachidonic acid recovered using the method of the present invention canbe used to test putative inhibitory agents in vitro by incubating theputative inhibitor with arachidonic acid under suitable conditionswell-known to those of skill in the art, and measuring leukotrieneproduction.

The following examples and test results are provided for the purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

This example describes the production of arachidonic acid by the strainS12 of Mortierella sect. schmuckeri which is a strain of Mortierellaschmuckeri.

A strain of Mortierella schmuckeri was identified in accordance with themethod of the present invention. Such strain is referred to herein asstrain S12. A one centimeter squared portion of a Mortierella schmuckeristrain S12 was cut from a solid agar plate and placed in 100 ml aliquotsof medium containing 10 grams/liter (g/l) of corn steep liquor, 0.1 g/lCaCO₃, 0.1 g/l MgSO₄7H₂O, 0.5 g/l KH₂PO₄, 1 milliliter per liter (ml/l)PII metals (6.0 g Na₂EDTA; 0.24 g FeCl₃-6H₂O; 6.84 g H₃BO₃; 0.86 gMnCl₂-4H₂O; 0.133 g ZnSO₄-7H₂O; 0.026 g CoCl₂-6H₂O; 0.005 g NaMoO₄-2H₂O;0.002 g CuSO₄-5H₂O and 0.052 g NiSO₄-6H₂O; dissolved in 1 liter of waterand pH adjusted to 8.0), and 1 ml/l vitamin mix (100 mg/L thiamin; 500μg/L biotin and 500 μg/L vitamin B₁₂), contained in 250 ml baffled shakeflasks. The cultures were incubated for 72 hours, at 30° C. on a rotaryshaker (225 rpm). After 72 hours, the cultures were of high density andhad stopped growing.

The cells in the flasks were then sampled to determine ash-free dryweights and to quantify the fatty acid content of the cells. The cellsof Mortierella schmuckeri strain S12 were harvested and centrifuged.Fatty acids in dry biomass of harvested cells were then methylated in 4%methanolic H₂SO₄ (4 ml H₂SO₄ in 96 ml methanol) at 100° C. for 1 hour.The fatty acid methyl esters were then quantified by gas chromatography(Varian 3500 gas chromatograph, Supelco SP 2330 column; initial columntemp. =70° C.; detector temp=250° C.; injector temp=220° C.; carrier gas=helium; temperature program: initial column temp =70° C. for 3 min, 20°C. per min to 195° C., then hold for 5 min., then 25° C. per min to 220°C., then hold for 8 min.). The composition of the fatty acids in thefungal biomass is shown in Table 1. TABLE 1 S12 Fatty Acid Profile FattyAcid Content FATTY ACID mg/g dwt* % TFA* MYRISTATE C14:0 0.9 0.3MYRISTOLEATE C14:1 1.1 0.3 PALMITATE C16:0 45.6 13.5 PALMITOLEATE C16:12.2 0.6 STEARATE C18:0 26.9 8.0 OLEATE C18:1 39.8 11.8 LINOLEATE C18:2N636.5 10.8 Gamma-LINOLENATE C18:3N6 13.8 4.1 LINOLENATE C18:3N3 1.6 0.5EICOSENOATE-11 C20:1 1.1 0.3 EICOSADIENOATE-11,14 C20:2 2.2 0.6HOMOGAMMA LINOLENATE C20:3N6 1.6 0.5 BEHENATE C22:0 17.4 5.2ARACHIDONATE C20:4 135.7 40.3 LIGNOCERATE C24:0 9.6 2.8 NERVONATE C24:10.6 0.2 336.5 100.0*TFA = Total Fatty Acids*dwt = cellular dry weight

The results indicated that under these fermentation conditions, thestrain S12 biomass contained about 33.7% of fatty acids. Approximately40.3% of the total fatty acids was comprised of arachidonic acid. Thearachidonic acid content of the biomass therefore was 13.6% cellular dryweight.

Example 2

This example describes the effect of varying the carbon to nitrogenratio in the fermentation medium on arachidonic acid production in thefermentation of Mortierella schmuckeri strain S12 cells.

Fermentation cultures were prepared as described in Example 1. Numerousfermentation samples were prepared that had increasing concentrations ofglucose (the amounts are shown in Table 2, first column). The relativeamounts of total fatty acids and arachidonic acid were measuredaccording to the method described in Example 1 and the results areillustrated in Table 2. TABLE 2 Strain S12: Effect of C:N Ratio on DryWeight, Lipid and Arachidonic Acid Yields Biomass Tot. Arachi- Arachi-glucose C:N dry wt. Final FA % donic % donic % g/L Ratio g/L pH dry wt.Tot. FA. dry wt. 3.7  3:1 3.1 7.3 15.9 48.6 7.7 6.2  5:1 4.0 7.1 24.043.4 10.4 12.4 10:1 6.0 6.6 31.3 35.5 11.1 37.2 30:1 6.2 6.5 31.5 35.611.2 49.6 40:1 6.6 6.5 32.0 37.5 12.0 74.4 60:1 6.8 6.4 30.9 39.3 12.199.2 80:1 5.8 6.4 25.7 37.8 9.7 124.0 100:1  5.9 6.4 25.5 37.4 9.5

The results indicated that optimal carbon to nitrogen ratio for thefermentation of strain S12 is about 40:1 to about 60:1. The results alsoindicate that the amount of arachidonic acid produced by S12 cells canbe increased by limiting the amount of non-carbon nutrients, inparticular nitrogen, in the fermentation medium.

Example 3

This example illustrates the effect of nutrient manipulation onarachidonic acid production by Mortierella schmuckeri strain S12 andMortierella camargensis strain S3.

A strain of Mortierella camargensis was identified in accordance withthe method of the present invention. Such strain is referred to hereinas strain S3. Fermentation cultures were prepared as described inExample 1. Numerous fermentation samples were prepared that haddifferent nutrients deleted from the fermentation medium. The nutrientsdeleted from the various fermentation samples are shown in Table 3(first column). The relative amounts of total fatty acids andarachidonic acid were measured according to the method described inExample 1. The results are shown in Table 3. TABLE 3 Strains S12 and S3:Evaluation of nutrient subtraction on ARA production Nutrient Biomassdwt Fatty acid % ARA % ARA % ARA Deleted yield g/L dwt TFA dwt g/LStrain S12: M. schmuckeri CaCO₃ 4.2 31.3 31.0 9.7 0.41 Vitamins 5.6 32.834.4 11.3 0.63 MgSO₄ 5.4 32.1 39.3 12.6 0.68 PII 5.5 30.6 34.3 10.5 0.58KH₂PO₄ 5.5 30.3 35.3 10.7 0.59 Strain S3: M. camargensis CaCO₃ 4.5 37.421.9 8.2 0.37 Vitamins 5.5 34.9 24.9 8.7 0.48 MgSO₄ 5.4 38.7 25.3 9.80.53 PII 5.5 34.9 23.2 8.1 0.45 KH₂PO₄ 5.3 34.1 23.2 7.9 0.42

The results indicated that, for both strains of Mortierella sect.schmuckeri, minimizing the magnesium concentration in the fermentationmedium had a greater effect on arachidonic acid production than deletionof calcium, vitamins, trace metals and potassium phosphate. For example,the amount of arachidonic acid produced by cells of strain S12 grown inthe absence of magnesium was about 0.7 grams of arachidonic acid perliter, while the arachidonic acid production by cells of strain S12grown in the absence of calcium was on average about 0.4 grams ofarachidonic acid per liter.

Example 4

This example describes a comparison of arachidonic acid production byMortierella camargensis strain S3 between cells grown in the presence orabsence of corn steep liquor, a complex nitrogen source.

A first fermentation sample was prepared using the method and culturemedium described in Example 1. A second fermentation sample was preparedusing the medium described in Example 1 but instead of corn steepliquor, yeast extract was used as the nitrogen source. Lipids wereprepared from strain S3 cells and analyzed using the method described inExample 1. The composition of the fatty acid mixture obtained from eachof the foregoing fermentation procedures is shown in Tables 4 and 5. TheS3 sample grown with corn steep liquor was found to contain 35.9% of dryweight as fatty acids. The arachidonic acid content of this sample was10.8% of cellular dry weight. The S3 sample grown without corn steepliquor was found to contain 19.8% of dry weight as of fatty acids. Thearachidonic acid content of the sample was 4.8% of cellular dry weight.TABLE 4 S3 Strain Grown With Corn Steep Liquor Fatty Acid Content FATTYACID mg/g dwt* % TFA* MYRISTATE C14:0 1.8 0.5 MYRISTOLEATE C14:1 0.9 0.3PALMITATE C16:0 60.1 16.7 PALMITOLEATE C16:1 1.0 0.3 STEARATE C18:0 30.38.4 OLEATE C18:1 27.9 7.8 LINOLEATE C18:2N6 51.4 14.3 GAMMA-LINOLENATEC18:3N6 27.5 7.7 EICOSENOATE-11 C20:1 1.5 0.4 EICOSADIENOATE-11,14 C20:23.1 0.9 HOMOGAMMA LINOLENATE C20:3N6 1.8 0.5 BEHENATE C22:0 28.2 7.8EICOSATRIENOATE C20:3 0.6 0.2 ARACHIDONATE C20:4 107.8 30.0EICOSAPENTANOATE C20:5N3 0.4 0.1 LIGNOCERATE C24:0 13.6 3.8 NERVONATEC24:1 0.8 0.2 DOCOSAHEXANOATE C22:6N3 0.6 0.2 359.4 100.0*TFA = Total Fatty Acids*dwt = cellular dry weight

TABLE 5 S3 Strain Grown Without Corn Steep Liquor Fatty Acid ContentFATTY ACID mg/g dwt* % TFA* MYRISTATE C14:0 1.0 0.5 MYRISTOLEATE C14:11.2 0.6 PALMITATE C16:0 38.9 19.7 PALMITOLEATE C16:1 0.8 0.4 STEARATEC18:0 9.7 4.9 OLEATE C18:1 33.6 17.0 LINOLEATE C18:2N6 28.1 14.2GAMMA-LINOLENATE C18:3N6 11.8 6.0 EICOSENOATE-11 C20:1 1.9 0.9EICOSADIENOATE-11,14 C20:2 1.0 0.5 HOMOGAMMA LINOLENATE C20:3N6 4.4 2.2BEHENATE C22:0 7.0 3.6 ARACHIDONATE C20:4 48.8 24.7 ERUCATE C22:1 0.00.0 EICOSAPENTANOATE C20:5N3 0.0 0.0 LIGNOCERATE C24:0 8.7 4.4 NERVONATEC24:1 0.4 0.2 DOCOSAHEXANOATE C22:6N3 0.4 0.2 197.7 100.0*TFA = Total Fatty Acids*dwt = cellular dry weight

The results indicated that inclusion of corn steep liquor as a nitrogensource in the fermentation medium enhanced arachidonic acid productionby S3 cells about two-fold. For example, S3 cells grown in the presenceof corn steep liquor produced about 107.8 milligrams of arachidonic acidper gram of fungal biomass. Arachidonic acid comprised about 30% of thetotal fatty acids. Conversely, S3 cells grown in the absence of cornsteep liquor produced about 48.8 milligrams of arachidonic acid per gramof fungal biomass. Arachidonic acid comprised about 24.7% of the totalfatty acids. Thus, fermentation in the presence of corn steep liquor (acomplex nitrogen source) enhanced the production of arachidonic acid.

The results indicate that corn steep liquor is one of the best complexnitrogen sources for stimulating arachidonic acid production in StrainS3. Arachidonic acid production, however, by Strain S12 (Mortierellaschmuckeri) is stimulated by a wider range of complex nitrogenincluding, but not limited to, corn steep liquor, yeast extract, yeast,whey and soy flour.

Example 5

This example describes a comparison between the arachidonic acid contentof Mortierella camargensis strain S3 and Mortierella schmuckeri strainS12 with previously known ATCC strains of the schmuckeri section ofMortierella.

Four strains, Mortierella camargensis strain S3, Mortierella schmuckeristrain S12 and Mortierella schmuckeri (ATCC No. 42658) were cultured inthe presence or absence of corn steep liquor as described in Example 4.The fatty acid content of the cells of the S3 strain and the two knownstrains was measured according to the method described in Example 1. Acomparison of the total fatty acid yield and arachidonic acid yields isshown below in Table 6. TABLE 6 Comparison of Arachidonic Acid and TotalFatty Acid Production in Strains of Mortierella from the SchmuckeriGroup of this Fungus w/o csl w/csl Total Fatty Acids (as % dwt)Mortierella camargensis (Strain S3) 19.8 35.9 Mortierella schmuckeri(Strain S12) 18.3 33.7 Mortierella schmuckeri (ATCC 42658) 28.2 38.0Arachidonic Acid (as % dwt) Mortierella camargensis (Strain S3) 4.9 10.7Mortierella schmuckeri (Strain S12) 6.0 13.6 Mortierella schmuckeri(ATCC 42658) 2.5 2.4 Arachidonic Acid (as % total fatty acids)Mortierella camargensis (Strain S3) 24.7 30.0 Mortierella schmuckeri(Strain S12) 32.6 40.3 Mortierella schmuckeri (ATCC 42658) 9.0 6.3w/o csl = without corn steep liquorw/csl = with corn steep liquor% dwt = percent dry weight of biomass

From the results shown in Table 6 it can be seen that the presence ofcorn steep liquor in the fermentation medium 30 increases the totalfatty acid production by about 2-fold in strain S3 and S12, and by aboutone third in Mortierella schmuckeri (ATCC No. 42658). However, while thepresence of corn steep liquor in the fermentation medium increasedarachidonic acid content (as % dwt) by about 2-fold in strain S3, thecorn steep liquor did not effect arachidonic acid production byMortierella schmuckeri (ATCC No. 42658). Mortierella clausenii (ATCC No.64864) showed no significant growth in either the presence or absence ofcorn steep liquor.

Example 6

This example describes the analysis of the fermentation productivity andlipid content of oil obtained from the Mortierella schmuckeri strainS12.

Fermentations were conducted using Mortierella schmuckeri strain S12 intwo 14 liter fermentation cultures designated vessel B20 and B23. M-3medium was utilized in vessel B20 and M-6 medium was utilized in vesselB23. M-3 medium contained 12 g/L Cargill 200/20 soy flour, 0.1 g/LMgSO₄-7H₂O, 0.1 g/L CaCO₃, 1 ml/L of PII Metals, 1 ml/l of Vitamin mix,2 g/L of KH₂PO₄, 43.8 g/L of glucose and 0.5 ml/L of K60K antifoam. M-6medium has the same ingredients as the M-3 medium except that itcontained 12 g/L of Nutrex 55 (Red Star Specialty Products, Milwaukee,Wis.), a spray dried form of inactive Bakers yeast, instead of soyflour. The oil samples were purified and analyzed for arachidonic acid(ARA) content. The results of these two fermentations are shown in Table7 below. TABLE 7 ARA Production by S12 cells from Large FermentationsApprox. Approx. Ferm. Vessel Biomass Yield ARA Yield Time ARA Prod. B2322 g/L 2.3 g/L 65.5 h 0.84 g/L/day B20 20 g/L 2.3 g/L 65.5 h 0.84g/L/day

Oil was extracted from the fungal biomass produced in the twofermentations according to the following process. Samples of S12fermentation broth from vessels B23 and B20 were filtered under vacuumto produce a cake of biomaterial which was isolated and dried in a steamoven. The dried biomaterial (200 g) was extracted with hexane (2×600 ml)in a waring blender to simulate a wet milling process. The milled fungalbiomass was filtered to remove solids and the hexane was evaporated toafford a crude oil. Approximately 75% of the theoretical oil content wasrecovered in this wet milling process. The crude oil was purified bypassing through a column of silica gel and the neutral oil fraction(triacylglycerides) was isolated by eluting the column with 30%ethanol:acetic acid in hexane. Fractions containing neutral oil (90% ofcrude oil) were pooled and concentrated to give a pure oil fractionwhich was analyzed by gas liquid chromatography. The results of thefatty acid analyses performed on the purified oil samples are shown inTable 8 below. TABLE 8 Fatty Acid Content of S12 Oil % FATTY ACID FattyAcid S-12 (B20) S-12 (B23) C16:0 Palmitate 10 11 C18:0 Stearate 12 12C18:1 n-9 Oleate 14 16 C18:2 n-6 Linoleate 10 9 C18:3 n-6 GLA 3 3 C20:0Arachidate 1 1 C20:3 n-6 Homo GLA 3 3 C20:4 n-6 Arachidonic Acid 41 37C22:0 Behenate 2 2 C24:0 Lignocerate 4 4

The results indicate that the purified oil obtained from biomassproduced in vessel B20 contained 41% arachidonic acid and the oil fromthe biomass produced in vessel B23 contained 37% arachidonic acid.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

1. A method to produce arachidonic acid, comprising culturingmicroorganisms of the genus Mortierella sect. schmuckeri in a mediumcomprising a source of assimilable organic carbon and a source ofassimilable nitrogen.
 2. The method of claim 1, wherein said Mortierellasect. schmuckeri is capable of producing at least about 0.70 grams ofarachidonic acid per liter per day.
 3. The method of claim 1, whereinsaid Mortierella sect. schmuckeri is capable of producing at least about0.80 grams of arachidonic acid per liter per day.
 4. The method of claim1, wherein said Mortierella sect. schmuckeri is capable of producing atleast about 0.86 grams of arachidonic acid per liter per day.
 5. Themethod of claim 1, wherein said method comprises culturing Mortierellasect. schmuckeri of the species Mortierella schmuckeri.
 6. The method ofclaim 1, wherein said method comprises culturing Mortierella sect.schmuckeri of the species Mortierella camargensis.
 7. The method ofclaim 1, wherein said Mortierella sect. schmuckeri is capable of growingas a dispersed filamentous form when grown under liquid cultureconditions.
 8. The method of claim 1, wherein said medium comprises acomplex nitrogen source.
 9. The method of claim 8, wherein saidcomponent increases arachidonic acid production by said Mortierellasect. schmuckeri by at least about 50 percent, as measured by percentcell dry weight or percent of total fatty acids in an oil, compared withMortierella sect. schmuckeri grown in the absence of said complexnitrogen source.
 10. The method of claim 8, wherein said componentincreases arachidonic acid production by said Mortierella sect.schmuckeri by at least about 100 percent, as measured by percent celldry weight or percent of total fatty acids in an oil, compared withMortierella sect. schmuckeri grown in the absence of said complexnitrogen source.
 11. The method of claim 1, further comprising limitinga non-carbon nutrient sufficiently to stimulate lipid production in saidmicroorganisms.
 12. The method of claim 11, wherein said non-carbonnutrient comprises nitrogen.
 13. The method of claim 1, wherein saidmethod further comprises recovering lipids comprising said arachidonicacid from said Mortierella sect. schmuckeri.
 14. A food product,comprising a microorganism of the genus Mortierella sect. schmuckeri anda food material.
 15. The food product of claim 14, wherein said foodmaterial comprises an animal food.
 16. A food product, comprising lipidsrecovered from a microorganism of the genus Mortierella sect. schmuckeriand a food material.
 17. The food product of claim 16, wherein saidMortierella sect. schmuckeri is capable of producing at least about 0.70grams of arachidonic acid per liter per day.
 18. The food product ofclaim 16, wherein said Mortierella sect. schmuckeri is of the speciesMortierella schmuckeri.
 19. The food product of claim 16, wherein saidMortierella sect. schmuckeri is of the species Mortierella camargensis.20. The food product of claim 16, wherein said food product has a totalfatty acid content in which up to about 20% by weight of total fattyacids is arachidonic acid.
 21. The food product of claim 16, whereinsaid food product has a total fatty acid content in which up to about10% by weight of total fatty acids is arachidonic acid.
 22. The foodproduct of claim 16, wherein said food product has a total fatty acidcontent in which between about 0.1% and about 1.0% by weight of totalfatty acids is arachidonic acid.
 23. The food product of claim 16,wherein said food material comprises an infant food material.
 24. Thefood product of claim 16, wherein said food material is selected fromthe group consisting of infant formula and baby food.
 25. A therapeuticagent, comprising lipids recovered from a microorganism of the genusMortierella sect. schmuckeri.
 26. The therapeutic agent of claim 25,wherein said agent is delivered by a medium selected from the groupconsisting of a capsule, a parenteral formulation and a liquid dietformulation.